Cells migrate by applying rearward forces against extracellular media. It is unclear how this is achieved in amoeboid migration, which lacks adhesions typical of lamellipodia-driven mesenchymal migration. To address this question, we developed optogenetically controlled models of lamellipodia-driven and amoeboid migration. On a two-dimensional surface, migration speeds in both modes were similar. However, when suspended in liquid, only amoeboid cells exhibited rapid migration accompanied by rearward membrane flow. These cells exhibited increased endocytosis at the back and membrane trafficking from back to front. Genetic or pharmacological perturbation of this polarized trafficking inhibited migration. The ratio of cell migration and membrane flow speeds matched the predicted value from a model where viscous forces tangential to the cell-liquid interface propel the cell forward. Since this mechanism does not require specific molecular interactions with the surrounding medium, it can facilitate amoeboid migration observed in diverse microenvironments during immune function and cancer metastasis.

The present model of G protein activation by G protein-coupled receptors exclusively localizes their activation and function to the plasma membrane (PM). Observation of the spatiotemporal response of G protein subunits in a living cell to receptor activation showed that 6 of the 12 members of the G protein ␥ subunit family translocate specifically from the PM to endomembranes. The ␥ subunits translocate as ␤␥ complexes, whereas the ␣ subunit is retained on the PM. Depending on the ␥ subunit, translocation occurs predominantly to the Golgi complex or the endoplasmic reticulum. The rate of translocation also varies with the ␥ subunit type. Different ␥ subunits, thus, confer distinct spatiotemporal properties to translocation. A striking relationship exists between the amino acid sequences of various ␥ subunits and their translocation properties. GPCR2 stimulation results in the activation of G protein ␣ and ␤␥ subunit complexes which modulate the function of downstream effector molecules that function on the cytosolic surface of the PM (1-4). The classic model of GPCR action, thus, restricts the activation of G proteins and consequently their effectors to the two-dimensional plane of the PM (1-4). Intracellular effects have been thought to occur through second messengers released through the activation of effector molecules such adenylyl cyclase, phospholipase C␤, and ion-conducting channels. Direct communication of a GPCR with intracellular membranes through the G protein subunits has not been anticipated. We have used live cell imaging methods to examine the spatiotemporal dynamics of the localization of components of a GPCR-mediated signaling pathway when the pathway is activated and deactivated.In mammalian cells similar to GPCRs the G protein subunits are also families of proteins. Based on the presence of distinct genes, there are 16 ␣ subunits, 5 ␤ subunit, and 12 ␥ subunit types. The ␣ subunit types appear to possess distinctly different properties (3, 4). Although evidence exists for the differential activity of ␤␥ subunit types in terms of their role in receptor activation of a G protein and modulation of effector function, these differences have been subtle and quantitative (5-12). One potential reason for a lack of evidence for qualitative differences in the properties of these diverse proteins is that most assays used so far to measure G protein function have used techniques that lead to disruption of cells. The possibility that these proteins are involved in spatially distinct functions has, thus, remained unexplored.Here we examined the entire family of ␥ subunit types complexed with different ␤ subunit types for potential translocation in response to GPCR activation in various cell lines. There have been previous indications from our laboratory that the ␤ 1 ␥ 11 and ␤ 1 ␥ 5 subunit complexes translocate away from the PM on receptor activation (13). To identify the mechanistic basis, the translocation properties were examined in the presence of inhibitors of vesicle-mediated trafficking, an acylati...

We have determined the relative abilities of several members of the G protein ␤ and ␥ subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian ␤1 and ␥3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for ␤-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of ␤ and ␥ subunit types varies widely. Third, the reporter activity evoked by a particular combination of ␤ and␥ subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid ␤␥ complex formed in the cell as determined by immunoprecipitation. These results suggest that different ␤ and ␥ subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all ␥ subunit types for the ␤1 subunit type explains the preponderence of this subunit type in most G proteins.Most of the neurohormonal signaling pathways in mammals are mediated by heterotrimeric G proteins (1, 2). Most cells contain many different G protein subunit types, and yet particular agonists evoke a highly specific response in a cell by activating a defined G protein-mediated signaling pathway (3). Various mechanisms can contribute to this specificity. For instance, in a cell that contains many different ␣, ␤, and ␥ subunit types, only certain types may be capable of forming a heterotrimeric complex because of the differences in the intrinsic affinity of these subunit types for one another. It has been shown that interactions between two different ␥ subunit types (␥1 and ␥2) and three different ␤ subunit types (␤1-␤3) are selective, indicating that this is indeed a mechanism for achieving specificity (4, 5). However, these experiments were performed using in vivo or in vitro systems that could detect differences in the ability of these subunit types to interact but were not sufficiently sensitive to detect low level interaction between some of the subunit types. To obtain a more sensitive measure of the interaction between various members of the ␤ and ␥ subunit families, we used the yeast two-hybrid system. In this system, the proteins with the potential to interact are expressed as hybrids of two different domains of a transcription factor. If the proteins interact with each other, the transcription factor domains are in proximity and capable of activating a promoter that controls reporter activity (6). We chose this system because it measures protein-protein interaction in a yeast cell and is therefore a reasonably accurate reflection of the ability of the interacting proteins to form a complex in a cell. Furthermore, it is highly sensitive in comparison with the methods used before to measure protein-protein interaction. For instance, in an assay of in...

Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors. Here we show that, after activation by receptors, the G protein ␤␥ complex selectively translocates to the Golgi. Receptor inactivation results in G␤␥ translocating back to the plasma membrane. Both translocation processes occur rapidly within seconds. The efficiency of translocation is influenced by the type of ␥ subunit present in the G protein. Distinctly different receptor types are capable of inducing the translocation. Receptor-mediated translocation of G␤␥ can spatially segregate G protein signaling activity.Heterotrimeric (␣␤␥) G proteins are localized to the plasma membrane of mammalian cells, facilitating interaction and activation by transmembrane G protein-coupled receptors (1-3). Extensive characterization of the effectors on which the G proteins act has suggested that the activated G protein ␣ and ␤␥ subunits function on the plasma membrane (3-6). It has been thought that the post-translational addition of a lipid moiety to the ␣ subunit and the ␥ subunit aids in the localization of G␣ and G␤␥ complex to the plasma membrane, where they act on the effector molecules (7). However, there is little information about the properties of these proteins or the signaling they mediate in intact live mammalian cells, because studies attempting to observe G protein function in living mammalian cells have been limited.To visualize the impact of receptor activation and inactivation on the spatial distribution of G protein subunits, we tagged G protein subunits with the yellow and cyan mutant forms of the green fluorescent protein, YFP 1 and CFP, respectively. The fusion proteins were expressed in mammalian cell lines and observed after activating overexpressed or endogenous receptors using fluorescence-based imaging methods. Although we have previously obtained evidence indicating the direct involvement of the G protein ␥ subunit in receptor interaction (8, 9), we examined the effect of receptor activation on ␣o-CFP, ␤1, and different ␥ subunit types tagged with YFP in Chinese hamster ovary (CHO) cells overexpressing M2 muscarinic receptors. We discovered that ␥-YFP translocated from the plasma membrane to the cell interior on receptor activation and translocated back to the plasma membrane on inactivation. The ␤ subunit co-translocated with the ␥ subunit. The rapidity of the translocation process and proportion of ␤␥ complex that translocated were dependent on the ␥ subunit type present in the expressed G protein. Experiments using a marker for the Golgi complex and a Golgi disrupting agent, brefeldin A, indicated that the ␤␥ complex translocates to the Golgi complex. The translocation was sensitive to G i/o -and G q -coupled receptor stimulation. Endogenous receptors also stimulated G␤␥ translocation. The translocation of the ␤␥ complex is selective, because ␣o-CFP or a chimeric ␣o-q-CFP that couples to G q -coupling receptors do not translocate from the plasma membrane in response to rece...

G-protein-coupled receptor (GPCR) activity gradients evoke important cell behavior but there is a dearth of methods to induce such asymmetric signaling in a cell. Here we achieved reversible, rapidly switchable patterns of spatiotemporally restricted GPCR activity in a single cell. We recruited properties of nonrhodopsin opsins-rapid deactivation, distinct spectral tuning, and resistance to bleachingto activate native Gi, Gq, or Gs signaling in selected regions of a cell. Optical inputs were designed to spatiotemporally control levels of second messengers, IP3, phosphatidylinositol (3,4,5)-triphosphate, and cAMP in a cell. Spectrally selective imaging was accomplished to simultaneously monitor optically evoked molecular and cellular response dynamics. We show that localized optical activation of an opsin-based trigger can induce neurite initiation, phosphatidylinositol (3,4,5)-triphosphate increase, and actin remodeling. Serial optical inputs to neurite tips can refashion early neuron differentiation. Methods here can be widely applied to program GPCRmediated cell behaviors.optogenetics | cell polarity G -protein-coupled receptors (GPCRs) initiate most of the signaling in metazoans and regulate a wide variety of cellular responses that include differentiation, migration, secretion, and contraction. Asymmetric activation of GPCR signaling activity in a cell is thought to play a critical role in varied processes such as cell polarization (1) and modulation of neuron function (2). There is still limited information about activation of signaling that is restricted in space and time across a single cell. An impediment is the lack of methods to continuously vary signal input to a single cell with high time resolution and precision to quantitate second messenger and cellular output from the same cell. A method that provides reversible, temporal control over GPCR activity in restricted regions of a single cell may help govern cell behavior and probe the cellular and molecular basis of single-cell responses.Here we used a set of light-triggered GPCRs, human color opsins, and related nonrhodopsin opsins, to achieve such confined GPCR activation in a single cell. In contrast to molecular gradients, an optical signal provides higher spatiotemporal control and it can be switched on or off or relocated almost instantaneously. We recruited the properties of color opsins to develop optical triggers that spatiotemporally confine signaling. These opsins deactivate rapidly and demonstrate relatively low sensitivity to light (3, 4). The deactivation characteristics curtail the diffusion of activated receptors across a cell and help localize receptor activation to selected regions of a cell. Low susceptibility to bleaching allows continuous reproducible activation. In addition, nonrhodopsin opsins selectively activate different G-protein types, suggesting that they can be individually used to regulate distinct second messengers (4, 5). Rhodopsin or its chimeric forms have been valuable for globally activating G proteins (6-8). Howev...